The blood-brain barrier (BBB) is a tight barrier formed by microvessels and capillaries that control the passage of nutrients, fluids, metabolic products, and drugs between the blood and the brain. Impaired function of the BBB is involved in a number of major pathologies afflicting the brain, such as Alzheimer's disease, multiple sclerosis, Parkinson's disease, brain manifestations of AIDS, stroke, and cancer. Although the neurotherapeutics sector is among the largest and fastest growing markets in the pharmaceutical industry, progress is currently impaired by the lack of in vitro assays that reliabl predict in vivo BBB permeability. None of the existing models adequately replicates the organotypic microenvironment of the BBB, in which brain endothelial cells (ECs), pericytes (PCs) and astrocytes (ACs) are arranged in a characteristic architecture. The proposed work utilizes organ-on-chip technology recently developed by Nortis, Inc. for creating 3D tissue microenvironments in disposable microfluidic chips. The chip design enables the integration of living, lumenally perfused microvasculature, making it suitable for studying barrier function. Strikingly, extensive preliminary data indicate that human brain ECs, PCs, and ACs have the capacity to self-assemble into a BBB-like architecture within the Nortis chip. This data will be leveraged to further develop and eventually commercialize BBB models of mouse and human. The objective of Phase I is to achieve a model that replicates critical BBB functions of the mouse brain. The mouse model will be developed and optimized for viability, structure, and function. Expression of tight-junction (TJ) proteins and the transporter P-glycoprotein, an important functional characteristic of the BBB, will be measured. Microvessel permeability will be assessed by perfusion with fluorescently labelled molecules (Aim 1). The model will then be challenged with the barrier-modulating compound lipopolysaccharide (LPS), and evaluated for associated changes in TJ protein expression, molecule permeability, and leukocyte transendothelial migration (Aim 2). During Phase II, the mouse BBB chip will be used to develop and qualify specific BBB assays, such as transferrin receptor transporter activity, BBB permeability challenge with LPS, and stimuli-induced leukocyte transmigration (Aim 1). Success criteria is an assay robustness of Z' ? 0.2. Aim 2 of Phase II is to develop a human BBB model. The human model will be optimized to recapitulate key structural and functional features of the BBB, including TJ formation, permeability, and transporter activity. To demonstrate utility, the model will be treated with LPS, mannitol, and angiotensin II and evaluated for associated changes in BBB structure and function. Each of these compounds has clinical relevance but acts by a different mechanism. Aim 3 is to qualify specific human BBB assays and establish relevance to clinical data. The products developed with support from this grant will significantly enhance progress in basic, translational, and clinical neuroscience research and will significantly advance therapy for numerous devastating diseases.